Retinoic Acid-Induced Neurogenesis using Mouse Embryonic Carcinoma Cells
Published: August 30, 2024
Abstract
Source: Leszczyński, P., et al., Neurogenesis Using P19 Embryonal Carcinoma Cells J. Vis. Exp. (2019)
This video demonstrates a simple technique for retinoic acid-induced neurogenesis in P19 mouse embryonic carcinoma cells. Retinoic acid activates transcription factors and triggers gene expression for neuronal differentiation.
Protocol
1. Aggregate Generation
- Add 5 µL of RA (retinoic acid) (1 mM stock dissolved in 99.8% ethanol, stored at -20 °C) to the 10 mL of Differentiation Medium and mix well (final concentration of 0.5 µM RA).
NOTE: RA is light-sensitive. A low concentration of ethanol does not affect cell differentiation. - Add 10 mL of Differentiation Medium (with RA) to the 100 mm non-treated culture dish (dedicated to suspension culture).
- Seed the 1 x 106 cells in the 100 mm dish (Dish surface area 56.5 cm2).
- Put the flask with cells into the incubator at 37 °C and 5% CO2 for 2 days to promote aggregate formation.
- After 2 days, exchange the Differentiation Medium. Aspirate medium containing aggregates using a 10 mL pipette and transfer to a 15 mL tube at RT.
- Allow the aggregates to settle by gravity for 1.5 min at RT.
- Discard the supernatant.
- Add a fresh 10 mL of Differentiation Medium with 0.5 µM RA using a 10 mL serological pipette.
CAUTION: Do not pipette the cell aggregates up and down. - Seed the aggregates into a new 100 mm non-treated culture dish (dedicated to suspension culture).
- Place the plate in the incubator (at 37 °C and 5% CO2) for 2 days.
2. Aggregates Dissociation
- Aspirate the cell aggregates using a 10 mL pipette.
- Transfer the aggregates to a 15 mL tube. Allow the cell aggregates to settle by gravity for 1.5 min.
- Remove the supernatant.
- Wash the aggregates with DMEM alone (serum- and antibiotic-free).
- Allow the cell aggregates to settle by gravity sedimentation for 1.5 min at RT.
- Aspirate the supernatant and add 2 mL of trypsin-EDTA (0.25%).
- Place the cell aggregates into a water bath (37 °C) for 10 min. Agitate the aggregates gently every 2 min by tapping with a hand.
- Stop the trypsinization process by adding 4 mL of Maintenance Medium.
- Pipette the aggregates up and down 20 times using a 1 mL pipette.
- Centrifuge cells for 5 min at 200 x g and RT.
- Remove the supernatant and resuspend the cell pellet in 5 mL of Maintenance Medium.
- Determine the cell number with a cell counter.
3. Plating Cells
- Add 3 mL per well of Maintenance Medium to a 6-well plate.
- Seed cells in the 6-well culture plate at a density of 0.5 x 106/well.
- Incubate at 37 °C with 5% CO2 concentration.
Declarações
The authors have nothing to disclose.
Materials
| DMEM high glucose (4.5 g/L) with L-glutamine | Lonza | BE12-604Q | |
| Ethanol 99.8% | Chempur | CHEM*613964202 | |
| Fetal Bovine Serum (FBS) | EURx | E5050-03 | |
| Penicillin/Streptomycin 10K/10K | Lonza | DE17-602E | |
| Phosphate Buffered Saline (PBS), 1x concentrated without calcium and magnesium ions | Lonza | BE17- 517Q | |
| Retinoic acid | Sigma- Aldrich | R2625-50MG | Dissolved in 99.8% ethanol; store in -20 °C up to 6 months |
| Trypsin 0.25% – EDTA in HBSS, without calcium and magnesium ions,with Phenol Red | Biosera | LM-T1720/500 | |
| 1 mL Serological Pipettes | Profilab | 515.01 | |
| 10 mL Serological Pipettes | Profilab | 515.1 | |
| 100 mm dish dedicated for suspension culture | Corning | C351029 | |
| 15 mL centrifuge tubes | Sigma- Aldrich | CLS430791-500EA | |
| 5 mL Serological Pipettes | Profilab | 515.05 | |
| 6-well plate | Corning | CLS3516 | |
| Cell culture flasks, surface area 25 cm square | Sigma- Aldrich | CLS430639-200EA |
Citar este artigo
Retinoic Acid-Induced Neurogenesis using Mouse Embryonic Carcinoma Cells. J. Vis. Exp. (Pending Publication), e22531, doi: (2024).